Plasmid

Part:BBa_K3482025

Designed by: Thierry Marti   Group: iGEM20_UNILausanne   (2020-10-23)
Revision as of 17:32, 26 October 2020 by Santosmoreno j (Talk | contribs) (→‎Characterization)


pLPT119 repressilator plasmid(Potvin-Trottier, 2016)

This plasmid codes for a synthetic circuit composed of the transcription factors tetR, cI and lacI. It also codes for a constitutively expressed CFP reporter gene, and the mVenus reporter gene under the control of pTetO1

Usage and Biology

The repressilator is a synthetic regulatory network of at least three genes in a plasmid that repress each other in a feedback loop. These genes are cyclically expressed one after the other in a loop at regular intervals over time. This property of cyclic expression of each gene allows us to produce our anti-cancer drug regularly for cancer treatment. For our system to work efficiently, the sponge plasmid is applied together with the repressilator to smooth out the observed oscillations by buffering excess proteins produced by the main repressilator.

pLPT119 has the same composition as the repressilator system present on the plasmid pLPT234, with one fluorescent reporter gene less (mKate2 and its corresponding promoter were removed). This is then a two-reporter system, of which one is constitutively expressed and one oscillates.

BBa K3482023 Reprisillator.png

Schematic representation of the repressilator plasmid.


All of the plasmids presented here were obtained and ordered from Addgene. They were all made available by the Professor Johan Paulson. All the plasmids come form this article Potvin-Trottier, L., Lord, N., Vinnicombe, G. et al. Synchronous long-term oscillations in a synthetic gene circuit. Nature 538, 514–517 (2016). https://doi.org/10.1038/nature19841.

Characterization

We transformed the pLPT119 repressilator plasmid with the pLPT41 sponge plasmid into E. coli Nissle to check whether we obtained oscillations. The two figures show the oscillatory expression of our reporter genes for 12 hours, with the sponge plasmid pLPT41 in both E.coli Nissle 1917 and DHL708, respectively.

BBa K3482023 pLPT119with41resultsEcoli.png

Fluorescence over time plot of E. coli Nissle 1917Δclb containing plasmids pLPT119 and pLPT41. The double lines for each reporter gene represent two biological replicates.

BBa K3482023 pLPT119with41resultsDHL.png

Fluorescence over time plot of E. coli DHL708 containing plasmids pLPT119 and pLPT41.


In the pLPT119 repressilator we only expected the mVenus (green) reporter gene to have an oscillatory expression pattern. CFP (blue) was under the regulatory control of a constitutive promoter which meant we expected the expression of the reporter gene to be constant and follow the change in OD. These results suggest that the pLPT119 repressilator and pLPT41 sponge plasmid work in both E. coli DHL708 and E. coli Nissle 1917Δclb


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 75
    Illegal EcoRI site found at 831
    Illegal EcoRI site found at 1000
    Illegal EcoRI site found at 1096
    Illegal EcoRI site found at 6309
    Illegal EcoRI site found at 7647
    Illegal EcoRI site found at 8508
    Illegal XbaI site found at 1855
    Illegal XbaI site found at 5546
    Illegal XbaI site found at 6511
    Illegal XbaI site found at 7842
    Illegal SpeI site found at 3870
    Illegal PstI site found at 837
    Illegal PstI site found at 6395
    Illegal PstI site found at 7726
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 75
    Illegal EcoRI site found at 831
    Illegal EcoRI site found at 1000
    Illegal EcoRI site found at 1096
    Illegal EcoRI site found at 6309
    Illegal EcoRI site found at 7647
    Illegal EcoRI site found at 8508
    Illegal SpeI site found at 3870
    Illegal PstI site found at 837
    Illegal PstI site found at 6395
    Illegal PstI site found at 7726
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 75
    Illegal EcoRI site found at 831
    Illegal EcoRI site found at 1000
    Illegal EcoRI site found at 1096
    Illegal EcoRI site found at 6309
    Illegal EcoRI site found at 7647
    Illegal EcoRI site found at 8508
    Illegal BamHI site found at 849
    Illegal BamHI site found at 1117
    Illegal BamHI site found at 5540
    Illegal XhoI site found at 8588
    Illegal XhoI site found at 8669
    Illegal XhoI site found at 8697
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 75
    Illegal EcoRI site found at 831
    Illegal EcoRI site found at 1000
    Illegal EcoRI site found at 1096
    Illegal EcoRI site found at 6309
    Illegal EcoRI site found at 7647
    Illegal EcoRI site found at 8508
    Illegal XbaI site found at 1855
    Illegal XbaI site found at 5546
    Illegal XbaI site found at 6511
    Illegal XbaI site found at 7842
    Illegal SpeI site found at 3870
    Illegal PstI site found at 837
    Illegal PstI site found at 6395
    Illegal PstI site found at 7726
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 75
    Illegal EcoRI site found at 831
    Illegal EcoRI site found at 1000
    Illegal EcoRI site found at 1096
    Illegal EcoRI site found at 6309
    Illegal EcoRI site found at 7647
    Illegal EcoRI site found at 8508
    Illegal XbaI site found at 1855
    Illegal XbaI site found at 5546
    Illegal XbaI site found at 6511
    Illegal XbaI site found at 7842
    Illegal SpeI site found at 3870
    Illegal PstI site found at 837
    Illegal PstI site found at 6395
    Illegal PstI site found at 7726
    Illegal NgoMIV site found at 5322
  • 1000
    COMPATIBLE WITH RFC[1000]


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